Transcreener ADP2 FP assay

Transcreener® assays rely on direct, highly-specific detection of nucleotides using antibodies that are able to differentiate between nucleotides on the basis of a single phosphate group. These assays can be used across entire families of nucleotide-dependent enzymes. The Transcreener assays use a homogenous, competitive immunoassay format in which the antibodies are paired with high affinity fluorescent tracers. Displacement of the tracer by the nucleotide being detected causes a change in its fluorescence properties.

The Transcreener® ADP2 FP assay from BellBrook Labs is an extremely sensitive ADP assay with a more sensitive antibody against ADP, yielding an excellent signal at less than or equal to 10% ATP consumption for a broad range of initial ATP concentrations (0.1-1,000 μM). The result is the ability to screen low ATP Km enzymes, and to use initial velocity enzyme kinetics at or below ATP Km concentrations, which leads to accurate inhibitor potencies and the ability to use less enzyme and substrate. Ratiometric, red-shifted fluorescence polarization output minimizes signal variability and reduces compound interference.

In step two the Transcreener® ADP2 Detection Mixture, which contains an ADP Alexa633 tracer bound to an anti-ADP antibody, is added. If there is enzymatic activity resulting in necessary ADP then the bound tracer is displaced by the ADP. The free tracer rotates quickly leading to a lower polarization value. If there is no free ADP because of no enzymatic activity, the tracer is still bound to the antibody. This whole construct rotates very slowly giving a higher polarization number. Therefore, ADP production leads to a decrease in fluorescence polarization.